Proteins Purification

Choosing The Right Column Chromatography For Proteins Purification

Proteins are considered the essential building blocks that provide our body with the essential amino acids and nutrition. They play a pivotal role in improving our immunity, regulation of enzymes, energy production, boosting our metabolism, improving muscle function, regulating various physiological processes, improving growth hormone, and cellular growth.

Proteins are also essential for DNA replication and repair, How is that you ask? Since proteins are the building blocks for various structural components, Collagen, for example, provides our body with high strength and structure to our skin, bones, and connective tissues, greatly improving their wear and tear as well as quality. 

Proteins come in various forms and combinations, thus it is essential to implement a protein purification process before use, so you can isolate a specific protein or group of proteins from a complex mixture. You can use and implement different column chromatography techniques to obtain higher levels of protein purification and also use the process to divide, separate, and collect different types of protein compounds from a mixture. 

So, which column chromatography technique is best used for proteins purification? There are a few chromatography techniques used to obtain high-quality protein separation, let us see a few of them below.

 Ion Exchange Chromatography (IEX) is one unique chromatography technique where the protein compounds are separated based on their charge for proteins purification. In the stationary column when the protein mixture is passed through a silica gel column, the adsorbent helps separate and purify the mixture of proteins. Once the solvent is passed through the stationary and mobile phase, the eluted output consists of fractions of desired proteins with the highest level of purity.

Since the column contains charged resin beads, the protein molecules react differently with them and get adsorbed at various levels in the column as they attract oppositely charged proteins.

Another effective column chromatography technique used for protein purification is  Size-Exclusion Chromatography (SEC), where the protein mixture is loaded in a column filled with a silica gel matrix, which is highly porous in nature. Thus, the larger compounds tend to pass through the column whereas, the smaller ones tend to get absorbed in the porous structure.

Hydrophobic Interaction Chromatography (HIC) is next where, a hydrophobic resin is used in the column chromatography process, to retain proteins based on their hydrophobicity. In this chromatography technique, the column is packed with a resin with a mix of hydrophobic groups, and then in the mobile phase, the mixture is passed through high salt concentration, where at the end, the proteins and separated, purified, and eluted leading to proteins purification.

In the Affinity Chromatography technique, the stationary phase contains a ligand/substance that specifically binds to the target protein you want to extract or fraction from the existing mixture. It is one of the best and most efficient protein purifying techniques, as it works on the principles of affinity of the molecules to the adsorbent used in the column. When the purified and targeted protein binds to the ligand, it is eluted out of the column in the purest form. This chromatography technique is considered to produce the highest levels of protein purification amongst other techniques.

Reversed-Phase Chromatography (RPC), helps synthesize and purify proteins that are more hydrophobic in nature. It also works well on separating and purifying lipids, small peptides, and other forms of hydrophobic proteins. RPC implements a nonpolar stationary phase, where the column is packed with a hydrophobic resin which adsorbs the hydrophobic protein in the column for separation and protein purification.

The choice of chromatography technique here truly depends on the specific properties of the target protein purification and the level of purification you want to achieve as the end goal.